ARTICLE Received 20 Sep 2013 | Accepted 16 Oct 2013 | Published 18 Nov 2013 DOI: 10.1038/ncomms3788 MEIOB exhibits single-stranded DNA-binding and exonuclease activities and is essential for meiotic recombination Mengcheng Luo1, Fang Yang1, N. Adrian Leu1, Jessica Landaiche2, Mary Ann Handel3, Ricardo Benavente4, Sophie La Salle2 & P. Jeremy Wang1 Meiotic recombination enables the reciprocal exchange of genetic material between parental homologous chromosomes, and ensures faithful chromosome segregation during meiosis in sexually reproducing organisms. This process relies on the complex interaction of DNA repair factors and many steps remain poorly understood in mammals. Here we report the identi- fication of MEIOB, a meiosis-specific protein, in a proteomics screen for novel meiotic chromatin-associated proteins in mice. MEIOB contains an OB domain with homology to one of the RPA1 OB folds. MEIOB binds to single-stranded DNA and exhibits 30–50 exonuclease activity. MEIOB forms a complex with RPA and with SPATA22, and these three proteins co-localize in foci that are associated with meiotic chromosomes. Strikingly, chromatin localization and stability of MEIOB depends on SPATA22 and vice versa. Meiob-null mouse mutants exhibit a failure in meiosis and sterility in both sexes. Our results suggest that MEIOB is required for meiotic recombination and chromosomal synapsis. 1 Center for Animal Transgenesis and Germ Cell Research, Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, 200E Old Vet, Philadelphia, Pennsylvania 19104, USA. 2 Department of Biochemistry, Chicago College of Osteopathic Medicine, Midwestern University, Downers Grove, Illinois 60515, USA. 3 The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA. 4 Department of Cell and Developmental Biology, Biocenter of the University of Wu¨rzburg, Am Hubland, Wu¨rzburg 97074, Germany. Correspondence and requests for materials should be addressed to P.J.W. (email: [email protected]). NATURE COMMUNICATIONS | 4:2788 | DOI: 10.1038/ncomms3788 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3788 eiosis, the hallmark of gametogenesis in sexually cells14, and from XXY* testes, which lack all germ cells and thus reproducing organisms, creates haploid germ cells from contain only somatic cells15. By subtracting these two data sets Mdiploid progenitors and ensures their genetic diversity from the P18 data set, with a stringent cutoff value of at least 3 through recombination. Physical links between homologous unique peptides in P18 but none in P6 and XXY*, we identified 51 chromosomes permit the formation of crossovers required for putative meiotic chromatin-associated proteins (Fig. 1d and allele recombination and also facilitate accurate chromosome Supplementary Table S1). segregation at the first meiotic cell division1,2. Meiotic Identification of previously known meiotic chromatin-asso- recombination is initiated at DNA double-strand breaks (DSBs) ciated proteins confirmed the validity of our approach: 13 of generated by SPO11 (ref. 3); these DSBs become resected from 51 proteins identified are known to localize to meiotic the 50-end, yielding extended 30-single-stranded DNA (ssDNA) chromatin (SYCP1, SYCP2, SYCP3, SYCE2, SMC1B, HOR- overhangs4. Replication protein A (RPA) coats ssDNA, forming MAD1, H2AX, MDC1, TERF1, TDP1, PIWIL1, PIWIL2 and nucleoprotein filaments that prevent degradation and secondary YBX2; Supplementary Table S1), including all three synaptone- structure formation. Subsequent invasion of the 30-single strand mal complex proteins. Furthermore, the genes encoding 19 into the duplex of the homologue is mediated by DMC1 and proteins identified in our screen had been subjected to targeted RAD51. Strand invasion causes the displacement of one strand deletion in mice: 11 mutants exhibited meiotic arrest, 2 showed of the homologue, resulting in D-loop formation5. The invading post-meiotic spermiogenic arrest, 4 displayed embryonic lethality 30-end primes new DNA synthesis, such that D-loop extension (with an unknown role in meiosis) and 2 were dispensable for towards the second end allows for the capture of the 30-ssDNA fertility. The identification of these known meiosis factors end of the other break (second end). End ligation leads to the validates our biochemical/proteomic approach and suggests formation of double Holliday junctions. Although early steps of that the other 32 proteins identified in our screen are likely to meiotic recombination (DSB formation, end resection, strand be novel meiotic chromatin-associated proteins (Supplementary invasion and D-loop formation) have been relatively well Table S1). characterized, the intermediate steps of this process, such as second-end capture, are poorly defined. MEIOB is a novel meiosis-specific protein. To test whether the RPA is a conserved ssDNA-binding heterotrimer complex of newly identified putative meiotic chromatin-associated proteins RPA1, RPA2 and RPA3 (ref. 6). The four oligonucleotide-binding indeed localize to meiotic chromatin and function in meiosis, we (OB) folds of RPA1 mediate ssDNA binding of the RPA focused on one of these uncharacterized proteins—MEIOB. complex7. RPA is essential for many aspects of DNA MEIOB (470 amino acids (aa)) is predicted to contain an OB fold metabolism, notably DNA replication, DNA repair and meiotic (aa 167–272) with limited homology to the ssDNA-binding recombination. During meiotic recombination, RPA binds to domain-B OB domain of RPA1 (Supplementary Fig. S1). MEIOB various ssDNA intermediates, including resected 30-ssDNA and homologues are present in mammals, chicken, fugu fish, Droso- the D-loop. Studies of mutant alleles of yeast RPA1 have phila and Arabidopsis. Western blot analyses using specific anti- demonstrated that RPA is required for meiotic recombination8. bodies raised against MEIOB confirmed its association with RPA is indispensible for mouse development; a homozygous chromatin; MEIOB was also detectable in the cytoplasmic and point mutation in Rpa1 causes early embryonic lethality9. soluble nuclear fractions of the testis (Fig. 1e). Although abun- Here we report the identification of meiosis-specific with OB dantly expressed in the testis, MEIOB was not detectable in domains (MEIOB), a meiosis-specific protein with homology to somatic tissues when examined by western blotting (Fig. 1f). one OB fold of RPA1, in a systematic proteomics screen for MEIOB localized predominantly to the nuclei of spermatocytes in meiotic chromatin-associated proteins. We find that mouse the testis (Supplementary Fig. S2). Therefore, MEIOB is a novel MEIOB has evolved to fulfill an essential function in meiosis. meiotic chromatin-associated protein. Results MEIOB co-localizes with RPA in foci on meiotic chromosomes. Proteomics screen for meiotic chromatin-associated proteins. Immunostaining of spread nuclei of spermatocytes showed that To systematically identify proteins that are associated with MEIOB formed discrete foci on chromosomes, foci that exhibited meiotic chromosomes, we isolated chromatin from postnatal day a dynamic distribution pattern during meiosis (Fig. 2a–j). MEIOB 18 (P18) testes, which lack post-meiotic germ cells and are thus foci were absent at the early leptotene stage (Fig. 2a), became first highly enriched for meiotic germ cells. Our adaptation of a detectable on late leptotene chromosomes (Fig. 2b), formed arrays chromatin purification protocol for testis samples yielded highly along synaptonemal complexes in zygotene and early pachytene pure chromatin (Fig. 1a)10,11. SYCP2, a synaptonemal complex spermatocytes (Fig. 2c,d), and disappeared in late pachytene protein associated with meiotic chromatin12, was present in the spermatocytes (Fig. 2e). At a range of 100–200 per cell, MEIOB chromatin fraction, whereas the cytoplasmic germ cell-specific foci peaked at the zygotene stage (Fig. 2k). Notably, MEIOB foci protein TEX19 was not detectable in the chromatin fraction by were formed along the partially synapsed sex chromosomes at the western blot analysis (Fig. 1b)13. To improve detection of proteins pachytene stage (Fig. 2d). In addition, MEIOB formed foci on with low abundance, we separated chromatin-associated proteins meiotic chromosomes in fetal oocytes (Supplementary Fig. S3), from P18 testes by SDS–polyacrylamide gel electrophoresis implying functional relevance of MEIOB for meiosis in both (PAGE) and analysed ten different size fractions by tandem sexes. MEIOB was absent from the adult ovary (Fig. 1f), in which mass spectrometry (MS/MS; Fig. 1c). A total of 1,300 proteins oocytes are at the diplotene stage. No focal staining was observed with at least one unique peptide were identified. in Meiob À / À spermatocytes with the antibody used for these In addition to meiotic cells, P18 testes contain mitotic germ analyses, demonstrating that the antibody is specific for MEIOB cells and somatic cells (Fig. 1d). To identify proteins only and does not crossreact with RPA1 (Supplementary Fig. S4a). associated with meiotic chromatin, we applied a subtractive We next evaluated the potential interaction of MEIOB with approach, excluding chromatin-associated proteins present in other meiotic recombination-related proteins and found that mitotic cells. Using the same biochemical/proteomics protocol, MEIOB co-localized with RPA, which forms foci on prophase I we identified chromatin-associated proteins from postnatal